In single molecule fluorescence studies, background emission from labeled substrates often restricts their concentrations to non-physiological nanomolar values. One approach to address this challenge is the use of zero-mode waveguides (ZMWs), nanoscale holes in a thin metal film that physically and optically confine the observation volume allowing much higher concentrations of fluorescent substrates. Standard fabrication of ZMWs utilizes slow and costly E-beam nano-lithography. Herein, ZMWs are made using a self-assembled mask of polystyrene microspheres, enabling fabrication of thousands of ZMWs in parallel without sophisticated equipment. Polystyrene 1 μm dia. microbeads self-assemble on a glass slide into a hexagonal array, forming a mask for the deposition of metallic posts in the inter-bead interstices. The width of those interstices (and subsequent posts) is adjusted within 100–300 nm by partially fusing the beads at the polystyrene glass transition temperature. The beads are dissolved in toluene, aluminum or gold cladding is deposited around the posts, and those are dissolved, leaving behind an array ZMWs. Parameter optimization and the performance of the ZMWs are presented. By using colloidal self-assembly, typical laboratories can make use of sub-wavelength ZMW technology avoiding the availability and expense of sophisticated clean-room environments and equipment.
Coupling of motor proteins within arrays drives muscle contraction, flagellar beating, chromosome segregation, and other biological processes. Current models of motor coupling invoke either direct mechanical linkage or protein crowding, which rely on short-range motor-motor interactions. In contrast, coupling mechanisms that act at longer length scales remain largely unexplored. Here we report that microtubules can physically couple motor movement in the absence of short-range interactions. The human kinesin-4 Kif4A changes the run-length and velocity of other motors on the same microtubule in the dilute binding limit, when 10-nm-sized motors are separated by microns. This effect does not depend on specific motor-motor interactions because similar changes in Kif4A motility are induced by kinesin-1 motors. A micron-scale attractive interaction potential between motors is sufficient to recreate the experimental results in a computational model. Unexpectedly, our theory suggests that long-range microtubule-mediated coupling not only affects binding kinetics but also motor mechanochemistry. Therefore, motors can sense and respond to motors bound several microns away on a microtubule. These results suggest a paradigm in which the microtubule lattice, rather than being merely a passive track, is a dynamic medium responsive to binding proteins to enable new forms of collective motor behavior.
Coupling of motor proteins within arrays drives muscle contraction, flagellar beating, chromosome segregation, and other biological processes. Current models of motor coupling invoke either direct mechanical linkage or protein crowding, which rely on short-range motor–motor interactions. In contrast, coupling mechanisms that act at longer length scales remain largely unexplored. Here we report that microtubules can physically couple motor movement in the absence of detectable short-range interactions. The human kinesin-4 Kif4A changes the run length and velocity of other motors on the same microtubule in the dilute binding limit, when approximately 10-nm–sized motors are much farther apart than the motor size. This effect does not depend on specific motor–motor interactions because similar changes in Kif4A motility are induced by kinesin-1 motors. A micrometer-scale attractive interaction potential between motors is sufficient to recreate the experimental results in a biophysical model. Unexpectedly, our theory suggests that long-range microtubule-mediated coupling affects not only binding kinetics but also motor mechanochemistry. Therefore, the model predicts that motors can sense and respond to motors bound several micrometers away on a microtubule. Our results are consistent with a paradigm in which long-range motor interactions along the microtubule enable additional forms of collective motor behavior, possibly due to changes in the microtubule lattice.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.